Constitutive expression of non-infectious HIV-like particles

ABSTRACT

Non-infectious, non-replicating immunogenic HIV-like particles are produced by stable long-term constitutive expression in mammalian cells by eliminating elements toxic to the mammalian cells. An expression vector contains a nucleic acid molecule comprising a modified HIV genome devoid of long terminal repeats and wherein Tat and vpr sequences are functionally disabled and a constitutive promoter operatively connected to the modified HIV genome for constitutive expression of the modified genome to produce the HIV-like particles.

REFERENCE TO RELATED APPLICATIONS

This application is a United States National Phase filing under 35 USC371 of PCT/CA98/01164 filed Dec. 14, 1998 which is a continuation ofU.S. patent application Ser. No. 08/991,773 filed Dec. 16, 1997 (nowU.S. Pat. No. 6,121,021.

FIELD OF INVENTION

This invention relates to the expression of non-infectious,non-replicating immunogenic HIV-like particles and, in particular, togenetic modifications required to obtain long term high levelconstitutive expression of such particles.

BACKGROUND TO THE INVENTION

Human immunodeficiency virus is a human retrovirus and is theetiological agent of acquired immunodeficiency syndrome (AIDS). It isestimated that more than 18 million people have been infected with HIVas of mid 1996 (ref. 1—various references are referred to in parenthesisto more fully describe the state of the art to which this inventionpertains. Full bibliographic information for each citation is found atthe end of the specification, immediately preceding the claims. Thedisclosure of these references are hereby incorporated by reference intothe present disclosure).

As the HIV-1 epidemic continues to spread world wide, the need for aneffective vaccine remains urgent. Efforts to develop such a vaccine havebeen hampered by three main factors: (a) the extraordinary ability ofthe virus to mutate; (b) inability of most known specificities ofanti-HIV antibodies to neutralise HIV primary isolates consistently; and(c) lack of understanding of the correlates of protective immunity toHIV infection. In view of the complex biology of HIV-host interactions,the most fruitful avenue may be development of multivalent HIVimmunogens tailored to HIV isolates in specific geographical locations.

CD8 CTL that kill HIV-infected cells and antibodies that broadlyneutralise HIV primary isolates might be protective anti-HIV immuneresponses in uninfected individuals who are subsequently exposed to HIV(ref. 2).

The definition of a successful preventative HIV immunogen iscontroversial. Protective anti-HIV immune responses may prevent HIVinfection completely, may allow only transient infection, leading toclearance of virus, or may merely limit the extent of HIV infection, butin so doing prevent the development of AIDS. One suggestion is thatclearance of HIV occasionally occurs after both maternal-fetal HIVtransmission (ref. 3) and sexual transmission of HIV (ref. 4).Consequently, if protective anti-HIV immune responses could be inducedby an immunogen in an HIV-uninfected person, protection might beachieved via early termination of HIV infection.

It has been shown that anti-recombinant (r) gp120 envelope antibodiesraised in animals or in human volunteers neutralise HIV grown inlaboratory-adapted T-cell lines but not primary isolates of the virusgrown in peripheral blood mononuclear cells. This observation raisesimportant questions about the roles of various spcificities ofneutralising antibodies in protection against HIV. The predominant typesof anti-HIV neutralising antibodies raised against gp120 are antibodiesagainst the third variable (V3) region of gp120, as well as antibodiesagainst the conformationally determined CD4 binding site centred aroundthe fourth constant (C4) region of gp120. Although laboratory-adaptedvariants are pathogenic and have caused AIDS in man after laboratoryaccidents (ref. 5), the relevance of these variants in vivo incommunity-acquired infections is unknown. Serum concentrations ofantibodies against the V3 gp120 region and of antibodies that neutraliselaboratory-adapted HIV strains do not protect individuals fromdeveloping AIDS (ref. 6), nor do anti-V3 antibodies seem protectiveagainst maternal-fetal HIV transmission (ref. 7).

Thus, for induction by HIV immunogens of neutralising antibodies toprevent HIV infection, HIV immunogens are probably needed which arecapable of inducing anti-HIV antibodies that neutralise both HIVlaboratory-adapted isolates and HIV primary isolates grown in peripheralblood mononuclear cells (ref. 8).

There is suggestive evidence that envelope oligomers of HIV primaryisolates may be appropriate immunogens for induction of anti-HIVneutralising antibodies against primary HIV isolates grown in peripheralblood mononuclear cells. Future studies are expected to focus on theenvelope of HIV primary isolates as the target of neutralisingantibodies. If HIV envelope oligomers are successful in inducingantibodies that neutralise HIV primary isolates, the neutralisingantibody specificity may be variant specific and, if so, the issue ofHIV variability would still need to be addressed.

Several candidate vaccines, based on different concepts, are atdifferent stages in the HIV vaccine development pipeline. Candidatevaccines based on the subunit recombinant envelope concept and producedin mammalian cells have been shown to protect chimpanzees from HIV-1infection and to be safe and reasonably immunogenic in humans, inducingneutralizing antibodies. A second generation of candidate vaccines,which are based on live vectors expressing the envelope and other HIV-1genes, and which are capable of inducing CTLs are beginning to beevaluated in human trials. Newer generations of candidate vaccines nowbeing mostly explored in animal experiments are using combinations ofsubunit recombinant proteins or live vectored vaccines with otherimmunogens, such as synthetic peptides or pseudovirions, or are based onmore novel approaches, including nucleic acid immunization and perhapswhole-inactivated or live attenuated vaccines.

However, there is a clear need for immunogenic preparationsincorporating antigens or antigen fragments from primary or clinical HIVisolates. These preparations will be useful as vaccine candidates, asantigens in diagnostic assays and kits and for the generation ofimmunological reagents for diagnosis of HIV and other retroviral diseaseand infection.

Particular prior art immunogenic preparations include non-infectious,non-replicating HIV-like particles. PCT applications WO 93/20220published Oct. 14, 1993 and WO 91/05860 published May 2, 1990 (WhiteheadInstitute for Biomedical Research), teach constructs comprising HIVgenomes having an alteration in a nucleotide sequence which is criticalfor genomic RNA packaging, and the production of non-infectiousimmunogenic HIV particles produced by expression of these constructs inmammalian cells.

PCT application WO 91/07425 published May 30, 1991 (Oncogen LimitedPartnership) teaches non-replicating retroviral particles produced bycoexpression of mature retroviral core and envelope structural proteinssuch that the expressed retroviral proteins assemble into buddingretroviral particles. A particular non-replicating HIV-1 like particlewas made by coinfecting mammalian host cells with a recombinant vacciniavirus carrying the HIV-1 gag and protease genes and a recombinantvaccinia virus carrying the HIV-1 env gene.

In published PCT application WO 91/05864 in the name of the assigneehereof (which is incorporated herein by reference thereto), andcorresponding granted U.S. Pat. Nos. 5,439,809 and 5,571,712, there isdescribed particular non-infectious non-replicating retrovirus-likeparticles containing at least gag, pol and env proteins in their naturalconformation and encoded by a modified retroviral genome deficient inlong terminal repeats and containing gag, pol and env genes in theirnatural genomic arrangement.

In WO 96/06177 and corresponding copending U.S. patent application Ser.No. 08/292,967 filed Aug. 22, 1994 (abandoned), assigned to the assigneehereof and the disclosures of which are incorporated herein byreference, there are described further mutations to the HIV genome ofthe constructs of U.S. Pat. Nos. 5,439,809 and 5,571,712 to reducegag-dependent RNA packaging of the HIV-1 genome, to eliminate reversetranscriptase activity of the pol gene product, to eliminate integraseactivity of the pol gene product and to eliminate RNAse activity of thepol gene product, through genetic manipulation of the gag and pol genes.

In the preferred vectors described in the aforementioned U.S. Pat. Nos.5,439,809 and 5,571,712 and U.S. application Ser. No. 08/292,967,abandoned, a metallothionein promoter is employed, which requires theaddition of an inducer for expression to be effected. The use of suchpromoters for commercial scale production of such HIV-like particles isimpractical, in view of the cost of the heavy metals employed and thetoxic effect of such heavy metals on the expression cells.

It is desirable, therefore, to employ a constitutive promoter forexpression of the HIV-like particles. However, it has been found thatsubstitution of a constitutive promoter, results in cell toxicity,limiting the useful period of induction of the HIV-like particles.

SUMMARY OF INVENTION

It has now been surprisingly found that, by effecting specific geneticmodification to the HIV genome, as set forth herein, it is possible toeffect long term constitutive expression of non-infectious,non-replicating, immunogenic HIV-like particles without causing anytoxic effect on the mammalian cells expressing the particles.

In accordance with one aspect of the present invention, there isprovided a nucleic acid molecule, comprising a modified HIV genomedevoid of long terminal repeats and wherein vpr and tat sequences arefunctionally disabled and a constitutive promoter operatively connectedto the modified HIV genome for constitutive expression of the modifiedgenome to produce non-infectious, non-replicating and immunogenicHIV-like particles.

The vpr and tat sequences may be functionally disabled by the insertionof stop codons therein preventing expression of the respective encodedgene products.

The HIV genome may be further modified by replacing the signal peptideof gp120 by a gp120 expression enhancing sequence, specifically thesignal peptide encoding sequence of glycoprotein D (gD) of herpessimplex virus (HSV).

In a preferred embodiment of the invention, the env gene in the nucleicacid molecule encodes a env gene product from a primary HIV-1 isolate.

In accordance with the aforementioned U.S. application Ser. No.08/292,967, abandoned, (WO 96/06177), the HIV genome of the nucleic acidmolecule may be further modified to effect reduction in gag-dependentRNA packaging of the gag gene product. Such reduction in gag-dependentRNA packaging of the gag gene product may be effected by replacing Cys395 and Cys 395 of the gag gene product of HIV-1 LAI isolate, or thecorresponding amino acids of another isolate, by serine.

In addition or alternatively, also in accordance with U.S. applicationSer. No. 08/292,967, abandoned, (WO 96/06177), the HIV genome of thenucleic acid molecule may be further modified to substantially eliminatereverse transcriptase activity, integrase activity and RNAse activity.In this regard, a BalI-BalI portion of the pol gene may be deletedbetween nucleotides 2655 and 4507 of the LAI isolate of HIV-1 or thecorresponding portion of the pol gene of another HIV-1 isolate.

The constitutive promoter employed herein may be the human immediateearly cytomegalovirus promoter or any other convenient constitutivepromoter of expression of the non-infectious, non-replicatingimmunogenic HIV-like particles in mammalian cells. An expressionenhancing sequence may be provided between the promoter and the modifiedgenomes. Such an expression enhancing sequence may be the humancytomegalovirus Intron A sequence.

For the purposes of expression of the HIV-like particles, the nucleicacid molecule provided herein may be incorporated into an expressionvector, which may be one having the identifying characteristics ofplasmid pCMVgDtat⁻vpr⁻, as described in detail below.

The present invention, in another aspect thereof, provides a method ofobtaining a non-infectious, non-replicating, immunogenic HIV-likeparticle, which comprises incorporating into an expression vector anucleic acid molecule comprising a modified HIV genome devoid of longterminal repeats and wherein vpr and tat sequences are functionallydisabled and a constitutive promoter operatively connected to themodified HIV genome, introducing the expression vector into mammaliancells, and constitutively expressing the nucleic acid molecule in thecells to stably produce non-infectious, non-replicating, immunogenicHIV-like particles.

The nucleic acid molecule incorporated into the expression vector mayhave the various features discussed above with respect to the nucleicacid molecule aspect of the invention. In addition to constitutiveexpression, expression of the nucleic acid molecule may also be enhancedby employing chemical agents which enhance the specific promoteremployed. The expression vector preferably is one having the identifyingcharacteristics of plasmid pCMVgDtat⁻vpr⁻.

Such method enables the HIV-like particles to be produced over a longterm without any adverse toxic effect on the mammalian cells. As seenbelow, applicants have maintained constitutive expression through morethan 50 passages of the cells. The present invention extends to thenon-infectious, non-replicating immunogenic HIV-like particles lackingTat and Vpr and producible by the method aspect of the invention as wellas immunogenic compositions comprising the same and methods ofimmunization using such compositions.

The non-infectious, non-replicating immunogenic HIV-like particlesproduced according to the procedure of the invention may be employed inimmunogenic compositions, as described in the aforementioned U.S. Pat.Nos. 5,439,809 and 5,571,712 and U.S. patent application Ser. No.08/292,967, abandoned (WO 96/06177), for inducing an immune response ina host.

Advantages of the present invention include the ability to effectlong-term production of non-infectious, non-replicating immunogenicHIV-like particles, thereby providing an expression system for suchparticles which is more useful in commercial production of the HIV-likeparticles than the systems previously considered.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the genetic map of expression plasmid p83-19 which containsa modified form of the 8.3 kb SacI-XhoI (nucleotides 678 to 8944) fromthe LAI HIV genome. The fragment lacks LTR elements and primer bindingsite and is inserted into an expression vector containing themetallothionein (MT) promoter and the SV40 virus polyadenylation site.The gag gene is modified to eliminate the RNA packaging sequences byreplacing the codons encoding the two cysteine residues (Cys 392 and Cys395) in the first Cys-His box by codons encoding serine. The pol genehas been modified by deletion of a portion to substantially remove thereverse transcriptase and integrase activities thereof. Aoligonucleotide has been inserted within the deleted pol gene tointroduce three stop codons in three different reading frames to preventthe remaining sequences of integrase from being translated. The env geneis a hybrid gene comprising the gp120 coding sequence of HIV-1 isolateMN and the gp41 coding sequence of isolate LAI.

FIG. 2A shows expression plasmid pCMVgDtat⁻vpr⁻ while FIG. 2B shows thegenetic map of the pCMV-pA segment of the expression plasmid. Theexpression plasmid pCMVgDtat⁻vpr⁻ is derived from plasmid p83-19(FIG. 1) and contains the human cytomegalovirus (CMV) promoter andenhancer element as well as CMV Intron A sequences in place of the MTpromoter. The coding sequences for the regulatory proteins Tat and Vprwere modified to prevent synthesis of both proteins upon expression. Thesignal peptide fragment of HIV-1 gp120 was replaced by the signalpeptide fragment of the glycoprotein D (gD) of Herpes Simplex Virus(HSV). In addition, the G418 resistance gene was co-linearly insertedinto the plasmid and placed under the regulation of the SV40 promoterand polyadenylation sequences.

FIG. 3, comprising panels A, B and C, shows Western blot analysis of HIVvirus-like particles expressed from a Vero cell line containing theexpression vector pCMVgDtat⁻vpr⁻ (clone Vero-356) which was establishedafter stable transfection of Vero cells. The Western blot analysis showsthe continued stable production of HIV-like particles with increasingpassage number, panels A (passages 14 to 18) and B (passages 46 to 51),and upregulation of particle production following induction with sodiumbutyrate (NaBu) alone or NaBu plus dexamethasone (Dexam), panel C.

GENERAL DESCRIPTION OF THE INVENTION

The present invention enables the long term constitutive expression ofnon-infectious, non-replicating immunogenic HIV-like particles to beachieved through genetic manipulation of the HIV-1 genome. As describedabove, the present invention uses a constitutive promoter which iscoupled to a modified HIV genome lacking LTRs.

The HIV genome is modified to disable vpr and tat to prevent theirexpression. Such disabling may be achieved by the insertion of stopcodons, including multiple stop codons, to prevent translation of thegenes and thereby preventing the formation of Vpr and Tat. By disablingthese genes and preventing their expression, any toxic effect of thesegene products on the mammalian cells is eliminated and hence a long-termproduction of the HIV-like particles can be achieved.

In addition, it is preferred to replace the sequence encoding theendogenous gp120 signal peptide with a signal peptide which enhancesexpression of the gp120, for example, the signal peptide of glycoproteinD of the Herpes Simplex Virus.

An expression vector useful herein may be prepared by geneticmodification of plasmid p83-19, shown in FIG. 1. This plasmid, thepreparation of which is described in the aforementioned U.S. patentapplication Ser. No. 08/292,967, abandoned (WO 96/06177), encodes anHIV-like particle deficient in a plurality of elements required forinfectivity and/or replication of HIV but dispensable for virus-likeparticle production. Plasmid p83-19 is derived from plasmid pMTHIVBRUdescribed in the aforementioned U.S. Pat. Nos. 5,439,809 and 5,571,712.The HIV-like particle contains the env gene product which issubstantially the envelope of HIV-1 isolate MN. The HIV-like particlemay contain other env gene products, particularly those from clinicalisolates from HIV-1 infected patients, such as a primary HIV-1 isolatefrom clades A, B, C, D, E and O, including the specific isolate Bx08.The env gene products also may be a chimer of the gp120 protein from onesource and the gp41 from another source, such as MN/LAI, Bx08/LAI andclades/LAI chimers.

In the plasmid p83-19, the HIV genome comprises the SacI-XhoIrestriction fragment of HIV-1 LAI isolate and encompasses nucleotides678 to 8944 and is deficient in primer binding site. The gag gene hasbeen modified to replace two cysteine residues (Cys³⁹² and Cys³⁹⁵) inthe gag gene product with serine, so as to inhibit RNA packaging.Furthermore, the pol gene has been modified to delete a large portion ofthe pol gene so as to remove the reverse transcriptase and integraseactivities of the pol gene product, with an oligonucleotide sequenceGTATAAGTGAGTAGCGGCCGCAC (SEQ ID NO:7) being inserted within the readingframes to introduce stop codons to prevent the remaining sequences ofintegrase from being translated.

The plasmid p83-19 is modified to provide plasmid pCMVgDtat⁻vpr⁻, aplasmid 14531 bp in length and the genetic elements and characteristicsof which are shown in FIGS. 2A and 2B. The human metallothionein (MT)promoter present in p83-19 is replaced by the human immediate earlycytomegalovirus (CMV) promoter and enhancer element. The signal peptideof gp120 is replaced by a sequence encoding the signal peptide fragmentof glycoprotein D (gD) of Herpes Simplex Virus (HSV). This replacementis achieved by site directed mutagenesis, as described in Example 2below, using specific primers.

The expression of the Tat protein is prevented by inserting stop codonsat an appropriate site in the tat gene, specifically employing two stopcodons (nucleotides TAATAG) replacing nucleotides TGGAAG (nucleotides5896 to 5901) of HIV-1_(LAI). Such mutation is effected by site directedmutagenesis, as described in Example 2 below, using a specific primer.

The expression of Vpr protein is prevented by inserting stop codons atan appropriate site in the vpr gene, specifically employing a stop codon(nucleotide TAG) at two different loci within the Vpr coding sequence,with the first stop codon replacing HIV-1_(LAI) nucleotides 5625 to 5627and the second stop codon replacing nucleotides 5631 to 5633. Suchmutation is effected by site-directed mutagenesis, as described inExample 2 below, using a specific primer.

A gene conferring resistance to G418 is colinearly inserted into theplasmid and placed under the regulation of SV40 promoter andpolyadenylation sequences. The final plasmid assembly is shown in FIG.2A.

The plasmid pCMVgDtat⁻vpr⁻ may be stably transfected into monkey kidneyVero cells or other mammalian cells by any convenient procedure forexpression of non-infectious, non-replicating immunogenic HIV-likeparticles therefrom. G418 resistance cell lines are cloned and screenedfor production of the particles in the culture supernatant by measuringthe amount of particle-associated Gag p24 protein using a suitableantibody. Such a screening procedure is described in the aforementionedU.S. Pat. Nos. 5,439,809 and 5,571,712.

Cell lines secreting the HIV-like particles were found to stably produceHIV-1-like particles and to continue to produce such HIV-like particleswith increasing passage number, as may be seen from the Western blotanalysis of FIG. 3. HIV-1-like particles can be isolated and analysed byWestern blot using monoclonal antibodies specific for p24 (Gag) andgp120 (Env).

The levels of expression of the HIV-like particle may be increased byinduction using sodium butyrate with or without dexamethasone, as seenfrom Example 3 and FIG. 3, panel C.

Since the genetic modifications which have been made to the HIV genomedo not involve modification to immunogenic components of the HIV-likeparticle, the immunogenicity of the particles, as shown in U.S. Pat.Nos. 5,571,712 and 5,439,809 and U.S. application Ser. No. 08/292,967,abandoned, (WO 96/06177), is not impaired.

The non-infectious, non-replicating immunogenic HIV-like particlesprovided herein can be used in a variety of ways, as described in moredetail below. The genetic modifications which have been made hereinenable such HIV-like particles to be produced on a commercial scale fromstably transformed cell lines expressing the particles in significantquantities, in contrast to prior art expression systems.

Vaccine Preparation and Use

One possible use of the non-infectious, non-replicating immunogenicHIV-like particles produced by the present invention is as the basis ofa potential vaccine against retroviral diseases including AIDS andAIDS-related conditions.

Immunogenic compositions, suitable to be used as vaccines, may beprepared from the non-infectious, retrovirus-like particles. Theimmunogenic composition elicits an immune response which producesantibodies that are antiviral. Should the vaccinated subject bechallenged by a retrovirus, such as HIV, the antibodies bind to thevirus and thereby inactivate it. The immunogenic composition may alsoelicit cytotoxic T-lymphocytes (CTLs) which are able to lysevirally-infected cells.

Vaccines may be prepared as injectables, as liquid solutions oremulsions. The non-infectious HIV-like particles may be mixed withpharmaceutically-acceptable excipients which are compatible with theretrovirus-like particles. Excipients may include water, saline,dextrose, glycerol, ethanol, and combinations thereof. The vaccine mayfurther contain auxiliary substances, such as wetting or emulsifyingagents, pH buffering agents, or adjuvants to enhance the effectivenessof the vaccines. Methods of achieving an adjuvant effect for the vaccineinclude the use of agents, such as aluminum hydroxide or phosphate(alum), commonly used as 0.05 to 0.1 percent solution in phosphatebuffered saline and other adjuvants, including QS21 and incompleteFreunds adjuvant. Vaccines may be administered parenterally, byinjection subcutaneously or intramuscularly. Alternatively, theimmunogenic compositions formed according to the present invention, maybe formulated and delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic composition may be administeredto mucosal surfaces by, for example, the nasal or oral (intragastric)routes. Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkalene glycols ortriglycerides. Oral formulations may include normally employedincipients, such as pharmaceutical grades of saccharine, cellulose andmagnesium carbonate. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained-release formulations orpowders and contain 10 to 95% of the retrovirus-like particles of theinvention.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as is therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and to produce acell-mediated immune response. Precise amounts of active ingredientrequired to be administered depend on the judgement of the practitioner.However, suitable dosage ranges are readily determinable by one skilledin the art and may be of the order of micrograms of the HIV-likeparticles. Suitable regimes for initial administration and booster dosesare also variable, but may include an initial administration followed bysubsequent administrations. One example of an immunization schedule isat least one primary immunization with an HIV-like particle, producedaccording to the present invention, followed by at least one secondaryimmunization with a synthetic tandem T-B peptide containing a HIV T-cellepitope and a HIV B-cell epitope as described in European Patent No. 0470 980 and corresponding copending U.S. patent application Ser. No.07/768,608 filed May 3, 1990, abandoned, or WO 94/29339 andcorresponding U.S. Pat. No. 5,639,854, assigned to the assignee hereofand the disclosures of which are incorporated herein by reference. Thedosage of the vaccine may also depend on the route of 30 administrationand will also vary according to the size of the host.

Molecules produced in accordance with the invention may further find usein the treatment (prophylactic or curative) of AIDS and relatedconditions, by acting either to displace the binding of the HIV virus tohuman or animal cells or by disturbing the 3-dimensional organization ofthe virus.

Immunoassays

The HIV-like particles produced by the method of the present inventionare useful as immunogens, as antigens in immunoassays includingenzyme-linked immunosorbent assays (ELISA), RIAs and other non-enzymelinked antibody binding assays, or procedures known in the art forscreening anti-retroviral compounds, for the detection ofanti-retroviral (for example, HIV) HIV antibodies and retroviral antigen(for example, HIV). In ELISA assays, the retrovirus-like particles areimmobilized onto a selected surface, for example a surface capable ofbinding proteins, such as the wells of a polystyrene microtitre plate.After washing to remove incompletely adsorbed retrovirus-like particles,a non-specific protein, such as a solution of bovine serum albumin (BSA)or casein, that is known to be antigenically neutral with regard to thetest sample may be bound to the selected surface. This allows forblocking of non-specific adsorption sites on the immobilizing surfaceand thus decreases the background caused by non-specific bindings ofantisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials to be tested, in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of BSA, bovine gammaglobulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sampleis then allowed to incubate for from about 2 to 4 hours, at temperaturessuch as of the order of about 25° to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution,such as PBS/Tween, or a borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound retrovirus-like particles, and subsequent washing, theoccurrence, and even amount, of immunocomplex formation may bedetermined by subjecting the immunocomplex to a second antibody havingspecificity for the first antibody. If the test sample is of humanorigin, the second antibody is an antibody having specificity for humanimmunoglobulins and in general IgG. To provide detecting means, thesecond antibody may have an associated activity, such as an enzymaticactivity that will generate, for example, a colour development uponincubating with an appropriate chromogenic substrate. Quantification maythen be achieved by measuring the degree of colour generation using, forexample, a visible spectra spectrophotometer.

In one diagnostic embodiment where it is desirable to identifyantibodies that recognize a plurality of HIV isolates, a plurality ofantigenically-distinct HIV-like particles of the present invention areimmobilized onto the selected surface. Alternatively, when the anti-HIVantibodies recognize epitopes that are highly conserved among variousHIV isolates (for example, a B-cell epitope from gag or gp41) a singleor a limited number of retrovirus-like particles may be immobilized. Ina further diagnostic embodiment where it is desirable to specificallyidentify antibodies that recognize a single HIV isolate (for example,LAI, MN, SF2 or HXB2), a single particular HIV-like particle of thepresent invention may be immobilized. This further diagnostic embodimenthas particular utility in the fields of medicine, clinical trials, lawand forensic science where it may be critical to determine theparticular HIV isolate that was responsible for the generation of animmune response including an antibody response.

In a further diagnostic embodiment, it may be desirable to specificallyidentify immunologically distinct retroviruses, for example, HIVisolates that belong to different clades. Immunologically distinct HIVisolates may include, for example, LAI, MN, SF2, HXB2 or a primary HIV-1isolate. In this diagnostic embodiment, a particular HIV-like particleof the present invention is useful for generating antibodies includingmonoclonal antibodies that specifically recognize such animmunologically-distinct HIV isolate.

It is understood that a mixture of immunologically distinct HIV-likeparticles may be used either as an immunogen in, for example, a vaccineor as a diagnostic agent. There may be circumstances where a mixture ofHIV-like particles are used to provide cross-isolate protection and/ordiagnosis. In this instance, the mixture of immunogens is commonlyreferred to as a “cocktail” preparation.

The present invention advantageously provides HIV-like particlescomprising gag and env gene products substantially in their nativeconformations. Such retrovirus particles will thus be recognized byconformational anti-HIV antibodies (such as anti-env antibodies) thatmay not recognize the HIV antigen in a denatured form or a syntheticpeptide corresponding to such an HIV antigen. The HIV-like particlesare, therefore, particularly useful as antigens and as immunogens in thegeneration of anti-retroviral antibodies (including monoclonalantibodies) in diagnostic embodiments.

Other Uses

Molecules which bind to the HIV-like particles, particularly antibodies,antibody-related molecules and structural analogs thereof, are also ofpossible use as agents in the treatment and diagnosis of AIDS andrelated conditions.

Variants of antibodies (including variants of antigen binding site),such as chimeric antibodies, humanized antibodies, veneered antibodies,and engineered antibodies that are specific for the retrovirus-likeparticles are included within the scope of the invention.

Antibodies and other molecules which bind to the HIV-like particles canbe used for therapeutic (prophylactic and curative) and diagnosticpurposes in a number of different ways, including the following:

For passive immunization by suitable administration of antibodies,possibly humanized antibodies, to HIV infected patients.

To activate complement or mediate antibody dependent cellularcytotoxicity (ADCC) by use of antibodies of suitable subclass or isotype(possibly obtained by appropriate antibody engineering) to be capable ofperforming the desired function.

For targeted delivery of toxins or other agents, for example, by use ofimmunotoxins comprising conjugates of antibody and a cytotoxic moiety,for binding directly or indirectly to cell-surface exposed HIV proteinsof HIV-infected cells (for example, gp120).

For targeted delivery of highly immunogenic materials to the surface ofHIV-infected cells, leading to possible ablation of such cells by eitherthe humoral or cellular immune system of the host.

For detection of HIV, using a variety of immunoassay techniques.

Thus, in yet a further diagnostic embodiment of the invention, theimmunogenic compositions (individually, or as mixtures includingcocktail preparations) are useful for the generation of HIV antigenspecific antibodies (including monoclonal antibodies) that can be usedto detect HIV or antigens, or neutralize HIV in samples includingbiological samples.

In an alternative diagnostic embodiment, the HIV-like particles can beused to specifically stimulate HIV specific T-cells in biologicalsamples from, for example, HIV-infected individuals for diagnosis ortherapy.

Biological Deposits

Certain plasmids that encode HIV-like particles and are employed inaspects of the present invention that are described and referred toherein have been deposited with the American Type Culture Collection(ATCC) located at 10801 University Boulevard, Manassas, Va. 20110-2209,USA, pursuant to the Budapest Treaty and prior to the filing of thisapplication. Samples of the deposited plasmids will become available tothe public upon grant of a patent based upon this United States patentapplication and all restrictions imposed on access to the deposit willbe removed. Deposits will be replaced if the depository is unable todispense viable samples. The invention described and claimed herein isnot to be limited in scope by the plasmids deposited, since thedeposited embodiment is intended only as an illustration of theinvention. Any equivalent or similar plasmids that encode similar orequivalent HIV-like particles as described in this application arewithin the scope of the invention.

Deposit Summary Plasmid ATCC Designation Date Deposited pMTHIVBRU 75852Aug. 4, 1994 pCMVgDtat⁻vpr⁻ 209446 Nov. 11, 1997

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitations.Immunological and recombinant DNA methods may not be explicitlydescribed in this disclosure but are well within the scope of thoseskilled in the art.

Example 1

This Example describes the construction of plasmid p83-19.

Plasmid p83-19 was constructed as described in the aforementioned U.S.patent application Ser. No. 08/292,967 (WO 96/06177) from pMTHIVBRU(ATCC 75852) as shown in FIG. 3 thereof. pMTHIVBRU is described in theaforementioned U.S. Pat. Nos. 5,439,809 and 5,571,712. Plasmid p83-19contains a hybrid envelope gene which was engineered by replacing DNAencoding most of gp120_(LAI) with the conjugate DNA encoding gp120_(MN).This result was accomplished by replacing a KpaI/BglII DNA fragment(nucleotides 6379 to 7668) from HIV-1_(LAI) with KpnI/BglII DNA fragment(nucleotides 6358 to 7641) from HIV-1_(MN). The genetic map for plasmidp83-19 is shown in FIG. 1.

Example 2

This Example describes the construction of plasmid pCMVgDtat⁻vpr⁻.

Plasmid pCMVgDtat⁻vpr⁻ was constructed from plasmid p83-19, (Example 1).The human metallothionein promoter from p83-19 was replaced with thehuman cytomegalovirus (CMV) promoter and enhancer element as well as CMVIntron A sequences. The CMV sequences that were used correspond to aSspI-PstI DNA fragment (nucleotides 460 to 2087) described in ref. 9.The signal peptide fragment from HIV-1 gp120 was replaced by the signalpeptide fragment of the glycoprotein D (gD) of Herpes Simplex Virus(HSV). This was accomplished by gene assembly-aided mutagenesis (GAAM),as previously described (ref. 10).

Three oligonucleotides were synthesized: an upstream primer having thesequence5′-TATGACGACAAACAAAATCACGGCCCCCAACCTGGCGGCAGTCCCCCCCATTGCCACTGTCTTCTGCTCTTTCTATTA-3′(SEQID NO: 1), in which the last 27 nucleotides are complementary tonucleotides 6230 to 6256 of HIV-1_(LAI), (all nucleotide numbering isaccording to ref. 11 and HIV Los Alamos Database, 1988); a downstreamprimer having the sequence5′-CCCATAATAGACTGTGACCCACAATTTTTCTGTGAGAGAGGCATCCGCCAAGGCATATTTGCCGCGGACCCCATGGAGGCCCAC-3′(SEQ ID NO:2), in which the first 33 nucleotides are complementary tonucleotides 6347 to 6379 of HIV-1_(LAI); a bridging oligonucleotidehaving the sequence 5′-TTGTTTGTCGTCATAGTGGGCCTCCATGGG-3′ (SEQ ID NO:3),in which the first 15 oligonucleotides are complementary to the 5′-end15 nucleotides of the upstream oligonucleotide while the last 15nucleotides are complementary to the 3′-end 15 nucleotides of thedownstream primer.

The expression of most of the Tat protein was prevented by inserting twostop codons (nucleotides TAATAG) which replaced nucleotides TGGAAG(nucleotides 5896 to 5901) of HIV-1_(LAI). This mutation was generatedby site-directed mutagenesis using the following oligonucleotide:5′-GACTTCCTGGATGCTATTAGGGCTCTAGTCTAG-3′ (SEQ ID NO:4). The expression ofmost of the Vpr protein was prevented by inserting a stop codon(nucleotides TAG) at two different loci within the Vpr coding sequences.The first stop codon replaced HIV-1_(LAI) nucleotides 5625 to 5627 whilethe second stop codon replaced nucleotides 5631 to 5633. These mutationswere inserted by site-directed mutagenesis using the followingoligonucleotide: 5′-AAGACCAAGGGCCATAGAGGTAGCCACACAATGAA-3′ (SEQ ID NO:5). Finally, the gene conferring resistance to G418 was co-linearlyinserted into the same plasmid and replaced under the regulation of theSV40 promoter and polyadenylation sequences. A map of the resultingplasmid pCMVgDtat⁻vpr⁻ is shown in FIG. 2A while details of the genomicmodifications are shown in FIG. 2B.

Example 3

This Example illustrates the constitutive expression of HIV-1-likeparticles from a Vero cell clone established after stable transfectionwith plasmid pCMVgDtat⁻vpr⁻.

Plasmid pCMVgDtat⁻vpr⁻ prepared as described in Example 2, was stablytransfected into monkey kidney Vero cells by the transfinity (BRL)calcium phosphate procedure. Approximately 400 stable G418^(R) celllines were cloned and screened for production of HIV-1-like particles bymeasuring the amount of particle-associated Gag p24 protein in theculture supernatants, as described in U.S. Pat. Nos. 5,439,809 and5,571,712. One cell line secreting about 50 ng/ml of p24 was identified(clone Vero-356) and found to stably produce HIV-1-like particles withincreasing passage number, as illustrated in FIG. 3, panels A and B.HIV-1-like particles were isolated by ultracentrifugation, and pelletedparticles were analysed by Western Blot as described in U.S. applicationSer. No. 08/292,967 (WO 96/06177) using monoclonal antibodies specificfor p24 (anti-p24) and gp120 (Mab 50.1) (FIG. 3, panels A and B). TheWestern blot analysis shows continued production of HIV-like particlesafter over fifty passages, in considerable contrast to the resultspreviously achieved.

The levels of particle production were increased three-fold by inducingclone Vero-356 with 5 mM sodium butyrate (NaBu), and five- to eight-foldby inducing the cell line with a mixture of 5 mM NaBu and 400 ng/mldexamethasone (Dexam) (FIG. 3, panel C).

SUMMARY OF THE DISCLOSURE

In summary of the disclosure, the present invention provides a novelprocedure for the preparation of non-infectious, non-replicatingimmunogenic HIV-like particles by constitutive expression by eliminatingelements which are toxic to the mammalian cells and nucleic acidmolecules useful in such procedure. Modifications are possible withinthe scope of the invention.

REFERENCES

1. World Health Organization, 1996. Wkly Epidemiol. Rec. 48:361.

2. Haynes B. F., Pantaleo G., Fauci A. S. 1996. Science 271:324-328.

3. Bryson Y. T., Pang S., Wei L. S. et al. 1995. N. Engl. J. Med.332:833-834.

4. Rowland-Jones s., Sutton J., Ariyosh K., et al. 1995. Nat. Med.1:59-64.

5. Pincus S., Messer K. G., Nara T. L., et al. 1994. J. Clin. Invest.93:2508-2513.

6. Hogervorst E., Jurrians S., de Wolf F., et al. 1995. J. Infect. Dis.171:811-821.

7. Markham R. B., Coberly J., Ruff A. J., et al. 1994. Lancet 343:1364.

8. Moore J. P. 1995. Nature 376:115.

9. Chapman, B. S. et al. 1991. Nucleic Acids Res. 19:3979-3986.

10. Yao F-L., Klein M. H., Loosmore S., Rovinski B. 1995. Bio Techniques18:372-376.

11. Wain-Hobson S., Sonigo P., Danos O., Cole S., Alizon M. 1985. Cell40:9-17.

7 1 78 DNA Artificial Sequence Description of Artificial SequenceSynthetic 1 tatgacgaca aacaaaatca cggcccccaa cctggcggca gtcccccccattgccactgt 60 cttctgctct ttctatta 78 2 84 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 2 cccataatag actgtgacccacaatttttc tgtgagagag gcatccgcca aggcatattt 60 gccgcggacc ccatggaggcccac 84 3 30 DNA Artificial Sequence Description of Artificial SequenceSynthetic 3 ttgtttgtcg tcatagtggg cctccatggg 30 4 33 DNA ArtificialSequence Description of Artificial Sequence Synthetic 4 gacttcctggatgctattag ggctctagtc tag 33 5 35 DNA Artificial Sequence Description ofArtificial Sequence Synthetic 5 aagaccaagg gccatagagg tagccacaca atgaa35 6 12 DNA Artificial Sequence Description of Artificial SequenceSynthetic 6 agtttcaata gt 12 7 23 DNA Artificial Sequence Description ofArtificial Sequence Synthetic 7 gtataagtga gtagcggccg cac 23

What we claim is:
 1. A nucleic acid molecule, comprising a modified HIV genome devoid of long terminal repeats and wherein vpr and tat sequences are functionally disabled by the insertion of stop codons therein preventing expression of the respective encoded gene products and the signal peptide encoding sequence of gp120 is replaced by the signal peptide encoding sequence of glycoprotein D of herpes simplex virus and a constitutive promoter operatively connected to said modified HIV genome for constitutive expression of said modified genome to produce non-infectious, non-replicating and immunogenic HIV-like particles.
 2. The nucleic acid molecule of claim 1 wherein an env gene of said modified HIV genome encodes an env gene product from a primary HIV-1 isolate.
 3. The nucleic acid molecule of claim 1 wherein said modified HIV genome is further modified to effect reduction in gag-dependent RNA packaging of the gag gene product.
 4. The nucleic acid molecule of claim 1 wherein said modified HIV genome is further modified to substantially eliminate reverse transcriptase activity, integrase activity and RNAse activity.
 5. The nucleic acid molecule of claim 1 wherein the constitutive promoter is the human immediate early cytomegalovirus promoter.
 6. An expression vector comprising the nucleic acid molecule of claim
 1. 7. The nucleic acid molecule of claim 3 wherein said reduction on gag-dependent RNA packaging of the gag gene product is effected by replacing Cys 392 and Cys 395 of the gag gene product of HIV-1 LAI isolate, or the corresponding amino acids of another HIV isolate, by serine.
 8. The nucleic acid molecule of claim 4 wherein a BalI-BalI portion of pol gene is deleted between nucleotides 2655 and 4507 of the LAI isolate of HIV-1 or the corresponding portion of the pol gene of another HIV-1 isolate.
 9. The nucleic acid molecule of claim 5 wherein an expression enhancing sequence is provided between said promoter and said modified genome.
 10. The nucleic acid molecule of claim 9 wherein said expression enhancing sequence is the human cytomegalovirus Intron A sequence.
 11. A method of obtaining a non-infectious, non-replicating, immunogenic HIV-like particle, which comprises: incorporating into an expression vector a nucleic acid molecule comprising a modified HIV genome devoid of long terminal repeats and wherein vpr and tat sequences re functionally disabled by the insertion of stop codon therein preventing expression of the respective encoded gene products and the signal peptide encoding sequence of gp120 is replaced by the signal peptide encoding sequence of glycoprotien D of herpes complex virus and a constitutive promoter operatively connected to said modified HIV genome, introducing the expression vector into mammalian cells, and constitutively expressing the nucleic acid molecule in said cells to stably produce non-infectious, non-replicating, immunogenic HIV-like particles.
 12. The method of claim 11 wherein the env gene encodes an env gene product from a primary HIV-1 isolate.
 13. The method of claim 11 wherein said HIV genome is further modified to effect reduction in gag-dependent RNA packaging of the gag gene product.
 14. The method of claim 11 wherein said HIV genome is further modified to substantially eliminate reverse transcriptase activity, integrase activity and RNAse activity.
 15. The method of claim 11 wherein the constitutive promoter is the human immediate early cytomegalovirus promoter.
 16. A non-infectious, non-replicating immunogenic HIV-like particle lacking Tat and Vpr and produced by the method of claim
 11. 17. The method of claim 13 wherein said reduction in gag-dependent RNA packaging of the gag gene product is effected by replacing Cys 392 and Cys 395 of the gag gene product of HIV-1 LAI isolate, or the corresponding amino acids of another HIV isolate, by serine.
 18. The method of claim 14 wherein a BalI-BalI portion of the pol gene is deleted between nucleotides 2655 and 4507 of the LAI isolate of HIV-1 or the corresponding portion of the pol gene of another HIV-1 isolate.
 19. The method of claim 15 wherein an expression enhancing sequence is provided between said promoter and said modified genome.
 20. The method of claim 19 wherein said expression enhancing sequence is the human cytomegalovirus Intron A sequence.
 21. The method of claim 20 wherein expression of the nucleic acid molecule also is induced.
 22. An immunogenic composition, comprising the non-infectious, non-replicating immunogenic HIV-like particle claimed in claim 16 and a physiologically-acceptable carrier therefor. 